Our long-term objectives are to understand the regulation of transcription and the combinatorial interactions of factors which serve to activate or silence gene expression. Regulation of the rate of transcription is a primary mechanism used in the cell to control the expression of endocrine hormones. Our objective is to develop immunological and biochemical strategies to delineate the combinatorial code which specifies expression of the insulin gene. Our general strategy is to investigate transcriptional regulators encoded by a single gene, the Pan gene. The Pan gene enodes the proteins Pan-1 and Pan-2, which are also known as E47 and E12. Pan-1 and 2 interact with enhancer elements present in genes and sets of genes selectively expressed in different tissues which include pancreatic, lymphoid and muscle tissues, and are therefore implicated as important regulators of processes as diverse as the control of metabolism, immune function and differentiation. Our studies will focus on Pan regulation of insulin gene expression. Four specific aims are proposed to investigate regulation of transcription by Pan proteins, using well characterized and conventional experimental approaches: 1. Generate antibodies that recognize Pan proteins and will distinguish Pan-1 and Pan-2. Mouse monoclonal antibodies and rabbit polyclonal antiseras will be generated against purified Pan proteins and peptide components of Pan proteins. These immunological reagents will be used to accomplish aims 2 and 3. 2. Characterize the presence and distribution of Pan proteins in vivo and in cell culture. The presence and cellular localization of Pan proteins in rat tissues and differentiated cell cultures will be investigated using antisera and monoclonal antibodies generated against Pan proteins with a particular focus on studies of the pancreas and pancreatic cell lines. 3. Characterize the components of complexes which interact with Pan binding enhancer elements. Protein extracts will be prepared from rat tissue and differentiated cell lines and assayed for components which interact with Pan binding enhancer elements. Biochemical and immunological strategies will be employed to resolve and characterize Pan element binding components. 4. Analyze the mechanism of transcriptional regulation by Pan-l and 2. Pan-1 and 2 regulatory function will be studied by site-directed mutagenesis and functional assays employing immunological reagents in simplified cell culture experiments and cell-free extracts.